A low-molecular-weight endoglucanase from Clostridium thermocellum similar to endoglucanase C. the specificity of effects on synthetic substrates and the amino acid composition

摘要

The substrate specificity, optimal activity, thermal stability, and amino acid composition of a low-molecular-weight cellulase of Clostridium thermocellum (35.5 kDa) isolated from the recombinant strain E. coli (pCU 104) were studied. The enzyme did not hydrolyze 3F-methylumbelliferyl (MUF)-#beta#-D-glucoside. However, it hydrolyzed 3F-MUF-cellobioside a maximum of the hydrolyzing activity at pH 5.3, although previous studies with a polymeric substrate showed that the enzyme had an optimum pH in the neutral range (6.0-7.5). The second-order rate constants (V/K_m) of the chromophore formation from chromogenic oligosaccharides increased with an increase in the length of the carbohydrate chain from 2 to 4 links, although the maximal rate (V) decreased in the same way. The heterosidic bond in 3F-MUF-#beta#-D-cellobioside was cleaved by the enzymes 15-30 times more often than the bond between two glucose residues. The optimal temperature for the enzyme activity at 57-62 deg C, the linearity of the Arrhenius plot in the range of 23-53 deg C, and the activation energy of 38.9 kJ/mol were determined. The half-inactivation time of the enzyme at 65 deg C was 36 min (pH 5.3). The amino acid composition of the endoglucanase was determined. It contained three cysteine residues. The content of proline in the purified protein was lower than in other cellulases of C. thermocellum.